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- W2057877158 abstract "We developed a near-field (NF) microscopy-based fluorescent correlation spectroscopy (FCS) approach that allows to measure protein and lipid mobility on living cells plasma membrane at sub-diffraction scale. The near-field excitation is obtained by means of an aluminum-coated optical fiber with sub-wavelength aperture (<100 nm in diameter), effectively reducing the illumination area of about one order of magnitude compared to standard confocal FCS. The use of this kind of probe also provides capability for dual-color FCS and fluorescence cross-correlation spectroscopy (FCCS) and guarantees the overlap of the excitation areas. The optical fiber is attached to an oscillating tuning fork and a shear force based feedback keeps the probe in the close proximity of the cell, preventing the membrane from fluctuating outside the evanescent field volume while minimizing probe-membrane interactions. We demonstrated the feasibility of the dual-color NF-FCS approach by measuring the diffusion of phosphoethanolamine or sphingomyelin simultaneously with GPI-anchored protein on the plasma membrane of living CHO cells. The comparison of these results with those obtained by confocal FCS highlights the advantages of the reduced illumination area in detecting anomalous or heterogeneous diffusion. Although other techniques have been shown to provide comparable illumination sizes, the NF-FCS approach offers the further advantage of dual-color FCS and FCCS and therefore represents a powerful tool to unravel the details of a variety of membrane processes occurring at the nanometric scale." @default.
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- W2057877158 date "2010-01-01" @default.
- W2057877158 modified "2023-10-18" @default.
- W2057877158 title "Near-Field Fluorescence Correlation Spectroscopy Approach to the Study of Living Cell Membrane Dynamics" @default.
- W2057877158 doi "https://doi.org/10.1016/j.bpj.2009.12.987" @default.
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